Embryo and Fingerling Toxicity of Dimethoate and Effect on Fecundity, Viability, Hatchability and Survival of Zebrafish, Danio rerio (Cyprinidae)

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1 World Journal of Fish and Marine Sciences 3 (2): , 2011 ISSN IDOSI Publications, 2011 Embryo and Fingerling Toxicity of Dimethoate and Effect on Fecundity, Viability, Hatchability and Survival of Zebrafish, Danio rerio (Cyprinidae) Shabnam Ansari and Badre Alam Ansari Department of Zoology, Zebrafish Laboratory, DDU Gorakhpur University, Gorakhpur (UP), India Abstract: Dimethoate, a broad spectrum organophosphate insecticide is a potential toxic pollutant, adversely affecting the fauna of aquatic ecosystem. In the present study, the adults, embryos and fingerlings of Zebrafish, Danio rerio were used as a model to investigate the toxic effects of Dimethoate. The 24 h to 96 h LC 10, LC 50 and LC 90 for the adults and fingerlings, slope and chi-square values were calculated. The 96 h LC50 value of Dimethoate was µg/l for adults. The 72 h LC50 for the embryo was µg/l and for the fingerlings the 96 h LC50 valueof Dimethoate was µg/l. The mortality of embryos at highest concentration i.e., 35 µg/l was increased to 88%. The adult fishes were exposed for one month to four different concentrations of Dimethoate (LC 5 to LC 20) and allowed to breed to observe the reproductive ability. The results show significant reductions in fecundity and hatchability in comparison to the control group. The survival of the hatched fingerlings was not affected after one week. The toxicity was concentration as well as time dependent. Key words: Danio rerio % LC % Dimethoate % Embryo % Fingerlings % Sub-lethal toxicity 50 INTRODUCTION the housefly and other household insects. Its mode of action is acetylcholinesterase (AChE) inhibition resulting Pesticides of various categories viz. organochlorines, to nerve exhaustion, nervous system failure and ultimately organophosphates, carbamates, synthetic pyrethroids to death. The signs of the toxicity of Dimethoate in fish, and natural products are used against a number of pests, Channa punctatus included jumping, erratic movement, to increase the crop production. These pesticides reach imbalance and death [12]. Frequently, organophosphorus the aquatic environment mainly by runoffs or drainage contamination has been found in environments, elements from treated agricultural lands, inadvertently exposing the of the food chain and humans [13]. non-target organisms especially the fish. They also Fish as a taxonomic group are the only primarily disrupt the food chain threatening the ecological balance aquatic vertebrate class and have, thus traditionally and the biodiversity of the nature. Aquatic contamination regarded as an indispensable component of integrated of these pesticides cause acute and chronic poisoning of toxicity testing strategies. Animal Welfare Organizations fish and cause severe damage to their vital organs [1, 2], have increasingly questioned ecotoxicity testing with fish skeletal deformities [3], reduced reproductive ability [4, 5] and stimulated efforts to develop various alternatives. A and causes various biochemical alterations [6, 7]. promising alternative approach to classical acute fish Recently, deltamethrin and neem-based pesticide achook toxicity testing with live fish is the fish embryo toxicity was found toxic to Zebrafish [8] and it is also reported test (FET) [14], which has been used for the exact that pyrethroid lambda-cyhalothrin and neemgold was evaluation of chemical toxicity to fish [15, 16]. The toxic to the adult and embryo of Zebrafish [9, 10]. Zebrafish, Danio rerio was selected for the present study Dimethoate is an organophosphorus pesticide with because they are model organisms for developmental a contact and systemic action, first described by toxicology research, readily available, produce large Hoegberg and Cassaday [11]. It was introduced in 1956 number of clear eggs and are sensitive to environmental and is produced in many countries for use against a broad changes. It is also recommended by International range of insects in agriculture and also for the control of Organization for Standardization [17] and the Organization Corresponding Author: Badre Alam Ansari, Department of Zoology, Zebrafish Laboratory, DDU Gorakhpur University, Gorakhpur (UP), India, Tel: , ba.ansari@rediffmail.com. 167

2 for Economic Co-operation and Development [18] for detected by their milky appearance and discarded. toxicological studies. The present study deals with the The hatched and dead embryos were recorded till 72 h and toxic effects of Dimethoate on the adult, embryo, survival of fingerlings up to one week was observed. fingerlings and reproductive ability of the Zebrafish. Embryos and Fingerlings Toxicity: For this study MATERIALS AND METHODS Zebrafish were bred in the laboratory to obtain the fertilized eggs. Five concentrations 15, 20, 25, 30 and 35 Zebrafish (Danio rerio) were reported from Uttar µg/l of Dimethoate for embryo and fingerling of Zebrafish Pradesh [19]. They were collected and acclimatized in the were selected with three replicates for each concentration. laboratory in 35 fingerling for each glass aquaria Stock solution was prepared by serial dilution of the containing dechlorinated water, aerated continuously pesticides in acetone. Acetone alone in the same amount through stone diffusers connected to a mechanical air served as control. Water was changed daily with fresh compressor. Water temperature ranged between 25±2ºC treatment of pesticide. and ph was maintained between Fish were fed For embryos toxicity tests lots of 100 fertilized eggs twice daily alternately with raw chopped goat liver and were separated in 500 ml glass beakers with 250 ml brine shrimps. Daphnia was also given as a live food dechlorinated water. Dead eggs were counted and oftenly as a supplement diet. removed daily until the end of the test. The dead embryos became white due to coagulation or precipitation of Adult Toxicity: For toxicity test adult Zebrafish of same protein. At the end of the incubation period (72 h) the age were procured from the general culture, for total hatched eggs were counted. determination of the 24, 48, 72 and 96 h LC 50 values using To determine the toxicity of Dimethoate for five different concentrations of Dimethoate 40, 60, 80, 100 fingerlings, 5-days old fingerlings were used. Three and 120 µg/l (Rogor 30% EC, Rallis India Ltd., Mumbai) replicates of ten fingerlings for each concentration were purchased from the local market. The pesticide was placed in 500 ml glass beakers having 250 ml of serially diluted in acetone. The tests were conducted in dechlorinated water. Five concentrations viz., 15, 20, dechlorinated water glass aquaria containing 10 25, 30 and 35 µg/l were selected. Mortality of fingerlings fingerlings for each. Two replicates of ten fishes for each was recorded after 24, 48, 72 and 96 h exposure periods. concentration of pesticide were performed. Randomization The susceptibility of the adults, embryos and fingerlings of the fish in the test aquaria was done according to the of Zebrafish to Dimethoate were established using the method prescribed by the U.S. Federal Water Pollution probit method of Finney [22] by StatPlus version Control Administration [20]. Water was changed daily 2009 computer software programme to calculate LC50 with fresh treatment of pesticide. A fish was considered values (with 95% confidence limits), slope and chi-square dead when its gill movements ceased and it did not values. respond to gentle prodding. Dead fishes was removed carefully from aquaria to avoid deterioration. RESULTS Reproductive Ability: For the study of reproductive The exposure of adult fishes to different ability, two months old adult matured fishes (10 females concentrations of pesticide showed abnormal behavioral and 20 males) were procured from the general stock and changes such as restlessness, aggregations at one corner exposed for one month to four different sub-lethal of the aquarium, erratic and jerky swimming, frequent concentrations of Dimethoate (96 h LC , LC , surfacing, increased mucous secretions and loss of LC and LC µg/l). Each set of experiment was balance. At high concentrations the pectoral and pelvic accompanied with a control group having no pesticide. fins were found to be expanded and they rolled vertically After one month continuous stress of the pesticide the prior to death. It is clear from table 1 that the mortality adult Zebrafish were brought back to the normal water for increases with the increase in concentrations and the LC50 breeding. Three matured females along with six males from decreases with the increase in the exposure period. It each test group were placed in 25-1 glass aquaria shows that toxicity of Dimethoate is concentration as well separately to breed in laboratory by the method of Ansari as time dependent. The 24 h LC 50 value of Dimethoate was and Kumar [21]. The eggs were counted and average µg/l which decreased to µg/l after 96 h of fecundity was established. Unfertilized eggs were exposure. The slope values shown in the table are steep. 168

3 Table 1: Toxicity of Dimethoate against Zebrafish. 95% Confidence limits of LC50 (µg/l) Treated Period (h) Effective Concentrations (µg/l) LCL UCL Slope Chi-square Values 24 LC LC LC LC LC LC LC LC LC LC LC LC Batches of ten fishes were exposed to five different concentrations of Dimethoate (diluted in acetone). Mortality was recorded every 24 h. Each set of experiment was replicated two times. The control groups were treated with acetone simultaneously. The LC50 values of the pesticide showed a significant (P<0.05) negative correlation with exposure time. LCL and UCL denote the lower and upper confidence limits respectively for the LC50 values. Table 2: Effect of Dimethoate on the fecundity, viability, hatchability and survival of Zebrafish, Danio rerio. Concentrations of Average number of Average number Survival of fingerlings Dimethoate of 96 h (µg/l) eggs laid/female of viable eggs Hatchability after 72 h after one week (96.38) 287 (980 LC (91.75) 258 (96.62) LC (84.35) 203 (91.85) LC (78.9) 162 (86.63) LC (53.59) 78 (80.41) Fishes were exposed to four different concentrations for one month under pesticidal stress of Dimethoate ranging from to µg/l. Data in parentheses are percent values. All the data were found significant (P<0.05) when Student s t-test was applied between treated and control. Table 3: Toxicity of Dimethoate to Zebrafish embryo and 5-day-old fingerlings. Fingerling toxicity Number of dead fingerlings Embryo toxicity Concentrations (µg/l) Number of dead embryos Total hatching in 72h 24h 48h 72h 96h 0 5 (1.67) 295 [98.30] NIL NIL NIL NIL (11.33) 266 [88.60] (26) 222 [74.00] (47) 159 [53.10] (69) 95 [31.50] (88) 36 [12.00] eggs were used in three batches of 100 each for embryo toxicity. 30 fingerlings were used in three batches of 10 each for fingerling toxicity. Data in parentheses ( ) shows the % mortality and [ ] shows the % hatching of the Zebrafish embryo. Table 4: Summary of probit analysis of Table 1. Effective Confidence limits Concentrations (µg/l) of LC50 (µg/l) Exposure` Test stage Duration (h) LC10 LC50 LC90 LCL UCL Slope Chi-square values Embryo Fingerling

4 The LC 50 values of the pesticide showed a significant workers [23, 24]. A significant decrease of AChE (P<0.05) negative correlation with the exposure time. The activity in Danio rerio adult specimens has been occurred chi-square values were not significant, indicating that the in chronic tests using 0.27 µg/l of parathion solution fish populations used in the experiments were within a Non Observed Effect Level (NOEL) of 0.12 µg/l homogeneous. [25]. It is also reported that AChE inhibition decrease the During the experiment a significant (P<0.05) reduction feeding rate due to impairment of impulse transmission in fecundity, viability and hatchability was observed [26]. (Table 2). It is revealed from the present experiment that Exemplifying [27] calculated the 96 h LC 50 values to the average number of eggs laid by Zebrafish is 320 under be 4.57 µg/l for Saccobrachus fossilis exposed to normal conditions whereas this number remarkably Dimethoate. The 96 h LC 50 values of Dimethoate for reduced to 224 after one month stress of LC 20 i.e catfish, Clarias batrachus was 65 mg/l [28]. In µg/l Dimethoate. A significant (P<0.05) decrease in Heteropneustes it was 2.98 mg/l [29]. It was reported hatchability up to 46.41% was observed. Results showed earlier that Zebrafish exposed to long term concentrations that, the survival of the hatched fingerlings was not of malathion, failed to spawn and showed skeletal affected after one week. deformities [3], inhibited acetylcholinesterase (AChE) The results of toxicity of Dimethoate to embryos and activity in the nervous tissue (Brain) of Zebrafish [30]. fingerlings of Zebrafish are illustrated in tables 3 and 4. During the present study 96 h LC 50 values of Dimethoate With increase in concentrations of pesticide the number for adult fishes was µg/l. Steep slope functions of of dead embryos in treated groups increased to 11.33% at the toxicity curves of 96 h mortality concentration data for 15 µg/l and at highest concentration i.e., 35 µg/l it was Dimethoate indicate a large increase in the mortality increased to 88% (Table 3). It is clear that at lowest associated with the relatively small increase in the concentration of pesticide the hatching was 266 (88.6%) concentration of this pesticide. This may be due to the which decrease to only 36 (12 %) at highest concentration rapid absorption of the pesticide and rapid onset of of pesticide as compared to the control group. Mortality effects [31]. of the control as well as treated embryos was observed The embryos and fingerlings toxicity tests are and the 72 h LC 50 value for embryo was calculated to be valuable for assessing potential impacts on growth, µg/l. At h, mortality of fingerlings was reproduction and survival of Zebrafish in polluted observed at different concentrations (15, 20, 25, 30 and 35 environment and are important tools for good µg/l). After exposure to the pesticide, the fingerlings of environmental monitoring [32, 33]. It has been observed Zebrafish also showed behavioral changes, they that increasing Dimethoate concentration had significant aggregated at one corner of the test aquarium, swimming effects on hatchability. Supporting this observation [34] fast at the water surface throughout the experiment. For who reported that early embryonic exposure in Medaka fingerlings the 24 h LC50 value of Dimethoate was was the greatest effect on hatching success. For example, µg/l, while for 48 h and 72 h it was µg/l and when embryos were exposed to 26 mg/l diazinon from µg/l respectively which decreased to µg/l after 96 h days 1-5, only 16% hatched whereas 100% hatched when exposure period (Table 4). The number of dead fingerlings exposed to the same concentration from days 5-9. At increases with increase in concentrations of pesticide. higher concentrations of pesticides the eggs of Cyprinus The results showed that, the effect of Dimethoate is carpio communis died before hatching because the concentration as well as time dependent. In this study the pesticides affects the activity of hatching enzymes [35]. slope functions also clearly indicate the acute toxicity of Sub-lethal effect of diazinon resulting AChE inhibition the pesticide. Thus, the results provide evidence that the could drastically affect growth, survival, feeding and Dimethoatepesticide is found to cause mortality of adults, reproductive behavior of fishes [36]. Moore and Wairing embryos and fingerlings of Zebrafish and it is not safe [37] and Wall [38] reported significant reduction in the which should be considered when used in agricultural levels of the reproductive steroids in Zebrafish (Danio areas near aquatic ecosystems. rerio) and Atlantic salmon (Salmo salar) after sub-lethal doses of diazinon. Hatchability was observed to be 30 and DISCUSSION 50% in eggs obtained from the mother fish exposed chronically to 2.6 and 1.3 mg/l for 30 days respectively During the present study, the erratic and abrupt [39]. In the present study, it can be attributed that the swimming in fishes after pesticide exposure may be due to hatching was affected due to the inhibition of some obstruction in AChE activity as suggested by other hatching enzymes. 170

5 During the development sensitivity may change with 3. Kumar, K. and B.A. Ansari, Malathion some compounds showing higher sensitivity in embryos Toxicity: Skeletal Deformities in Zebrafish whereas others are more toxic to larvae [40, 41]. Also, [15] found that, early life stages of Oryzias latipes were the most sensitive to toxic effect. Annune and Ajike [42] (Brachydanio rerio, Cyprinidae). Pestic. Sci., reported a very low LC 50 of mg/l for Oreochromis niloticus juveniles exposed to Dimethoate (Rogor). Oh et al., [43] present three factors causing the selective toxicity of diazinon for various fish species; different inhibition of AChE, different detoxification and absorption. During the present study there was a remarkable observation that the 72 h LC 50 for embryos and free swimming fingerlings were almost the same. This indicates that the chorion of the egg could not act as a barrier for Dimethoate and incapable to protect the embryo. This is in agreement with the finding of Scheil, et al., [44] who observed that the organophosphate, diazinon affected the embryo of Zebrafish and caused death due to circulatory failure. Several investigators have shown that organophosphates can penetrate the chorion and cause various teratogenic effects and death of fish embryos [45-47]. Very recently we also reported that the chorion of Zebrafish provides no protection to the developing embryo exposed to neem pesticides Neemgold and Azacel [9, 48]. It is concluded from the present study that Zebrafish and its early life stages are sensitive to low levels of Dimethoate in aquatic environment and significantly affect its populations. Therefore, these pesticides should be used with great caution and in a sustainable way so that it may not be hazardous to aquatic environment and human beings. Moreover, extensive investigations should be done for their safe use in aquaculture. ACKNOWLEDGEMENTS Authors are thankful to Prof. C. P. M. Tripathi, Head of the Department of Zoology, DDU Gorakhpur University, Gorakhpur for providing all the laboratory facilities to conduct this research work. REFERENCES 1. Omitoyin, B.O., E.K. Ajani, B.T. Adesina and C.N.F. Okuagu, Toxicity of lindane (Gamma Hexachloro-Cyclohexane) to Clarias gariepinus (Burchell 1822). World J. Zool., 1(1): Velmurugan, B.M., Selvanayagam, E.I. Cengiz and E. Unlu, The effects of monocrotophos to different tissues of fresh water fish, Cirrhinus mrigala. Bull. Environ. Contam. Toxicol., 78: : Sharma, D.K. and B.A. Ansari, Effect of the synthetic pyrethroid Deltamethrin and the neem based pesticide Achook on the reproductive ability of Zebrafish, Danio rerio (Cyprinidae). Arch. Pol. Fish., 18: Singh, P.K. and B.A. Ansari, Effect of Neem based formulations (Nimbecidine and Ultineem) on the spawning success of Zebrafish Danio rerio (Cyprinidae). The Bioscan, 5: Srivasatav, A.K., S.K. Srivastava and A.K. Srivastava, Response of serum calcium and inorganic phosphate of freshwater catfish, Heteropneustes fossilis to chlorpyrifos. Bull. Environ. Contam. Toxicol., 58: Mishra, D., S.K. Srivastava and A.K. Srivastava, Plasma calcium and inorganic phosphate levels of a teleost, Heteropneustes fossilis exposed to metacid-50. Malays. Appl. Biol., 33: Ansari, B.A. and D.K. Sharma, Toxic effect of synthetic pyrethroid Deltamethrin and Neem Based formulation Achook on Zebrafish, Danio rerio. Trends in Biosci., 2: Ansari, B.A. and M.K. Ahmad, 2010a. Toxicity of pyrethroid Lamba-cyhalothrin and Neemgold to the embryo of Zebrafish, Danio rerio (Cyprinidae). J. Appl. Biosci., 36: Ansari, B.A. and M.K. Ahmad, 2010b. Toxicity of synthetic pyrethroid Lambda-cyhalothrin and Neem based pesticides Neemgold on Zebrafish, Danio rerio (Cyprinidae). Global J. Environ. Res., 4: Hoegberg, E.I. and J.T. Cassaday, The reaction of O, O-dialkyl thiophosphoric acid salts some alpha-haloacyl derivatives. J. Am. Chem. Soc., 73: Dikshit, T.S.S. and R.B. Raizada, Toxicity evaluation of Dimethoate technical in fish (Report to Shaw Wallace and Co., India Ltd.). 13. Rodrigues, E.L., Alteracoes morfologicas em hepatocitos de Brachydanio rerio (Hamilton- Buchanan, 1822; 1823) exposto a acao do organofosfarado dimetoato 500 em dose subletal. Dissertacao de Mestrado, Universidade Federal do Parana, Curitiba, pp:

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